Degradation of TM₅₃₆ by ERAD. (A) His-tagged CS-TM₅₃₆ was overexpressed with inactive (−) or active (+) RHBDL2, soluble TM was purified by immobilized metal affinity chromatography from CM, and TM and HSPA5 were detected by immunoblot. (B) The TM-HSPA5 complex was detected by ELISA in the plasma from TM₅₃₆ patients (patients) and healthy participants (controls). (C) Cells overexpressing TM_WT or TM₅₃₆ were treated or not with MG (5 μM; 17 hours) then TM in cell lystates was detected by immunoblot. (D) Cells overexpressing TM₅₃₆ were treated with MG, cell lysates were deglycosylated with EndoH, and TM was detected by immunoblot. (E) TM detected by immunoblot in lysates of HUVEC and HUVEC₅₃₆ treated with MG (5 μM; 17 hours; GAPDH, loading control). (F) Cells overexpressing TM_WT or TM₅₃₆ were treated with CL (10 μM; 17 hours) and MG, and TM was detected by immunoblot of cell lysates. (G) Cells overexpressing TM_WT or TM₅₃₆ were treated with CL, and then TM in CM and cell lysates were measured by ELISA. Values are mean ± SD expressed as fold change compared with TM_WT without CL. Statistical analyses were performed using the Mann-Whitney test. ∗∗P < .01. Arrowheads on overexposed blots (OE) indicate bands that appear with MG132 treatment. MG, MG132.